The present invention relates to a firefly luciferase. More specifically, it relates to a firefly luciferase which emits light at high luminosity, the variants thereof, and a method for determining function of a cell by expressing a gene of the luciferase in a cell and detecting the light emission by means of imaging.
For determining function of cells such as intracellular signal transduction and gene expression, a fluorescent probe such as a fluorescent dye and fluorescent protein and a luminescent probe utilizing luciferine-luciferase reaction have been used. Especially, for the analysis of gene expression regulation, luminescence measurement which does not cause damage of cell due to exciton light or autofluorescence and is excellent in terms of quantitative determination is used. For example, in the case of observing a cell to which a luciferase gene is introduced, the intensity of expression of the luciferase gene (more specifically, the expression amount) can be determined by measurement of the luminescence from the cell by luciferase. The measurement of the luminescence is performed by the procedures in which luciferine, ATP, and the like are added to a lysate prepared by lysis of cells, and the solution is subjected to a quantitative determination by a luminometer utilizing a photoelectric multiplier. Namely, the degree of luminescence is measured after lysis of cells, and thus the expression amount of luciferase gene at a certain time point is determined as an average value of the entire cell. Examples of a method for introducing a luminescence gene such as luciferase gene as a reporter gene are a calcium phosphate method, lipofection, and electroporation, and each of these methods is used in accordance with the purpose and type of cells. Analysis of the expression amount of luciferase with use of an objective DNA fragment ligated to the upstream or downstream of luciferase gene to be introduced into a cell enables study of the effect of the DNA fragment on transcription of luciferase gene. Further, co-expression of luciferase gene to be introduced into a cell and the objective gene enables study of the effect of the gene product on expression of a luciferase gene.
For time-course analysis of the expression amount of a luminescence gene, the degree of luminescence of a living cell needs to be measured over time. Such measurement is carried out by cell cultivation in an incubator provided with a luminometer and quantitative determination of the degree of luminescence from the whole cell population for a given length of time. In this way, an expression rhythm etc. having a certain cycle can be analyzed, and the time course of the expression amount of the luminescence gene in the entire cell can be obtained.
In recent years, in biology and medical science fields there is increasing necessity of the time course observation of dynamic alterations in living samples with images. In a field of utilizing observation of fluorescence, time lapse or dynamic image pickup has been adopted for dynamically understanding function of a protein molecular. In the conventional technique, time course observation with use of a fluorescent sample has been carried out, for example, observation of moving images for one molecule of a protein provided with an added fluorescent molecule.
In contrast, when a luminescent sample is used for time-course observation, a CCD camera equipped with an image intensifier is required since the luminous intensity of the luminescent sample is extremely low. Recently, a microscope equipped with an optical system for observation of luminescent samples has been developed (Jpn. Pat. Appln. KOKAI Publication No. 2006-301599, International Publication No. 2006/088109).
Upon image pickup of a luminescent sample having small luminous intensity, it should be exposed for a longer term for obtaining clear image. Such a luminescent sample is used for only limited research. For example, when 30 minutes of exposure is required because of low luminous intensity, time-course image pickup is possible at every 30 minutes but is not at a shorter time, and real-time image pickup is also impossible. Plural images should be obtained and compared in order to focus on cells which emit light, and thus when longer exposure time is required because of low luminous intensity, it is time-consuming.